SUMMARY The functional repertoire of KCNQ1 and HERG channels on the cardiomyocyte sarcolemma is sustained by dynamic protein trafficking, sorting, and degradation processes. Reduced surface density of KCNQ1 and HERG is a major mechanism underlying LQT1 and LQT2, respectively, motivating a need to understand fundamental mechanisms regulating channel trafficking and stability. Posttranslational modifications by ubiquitin looms as a particularly powerful determinant of KCNQ1 and HERG channels as they can potentially regulate multiple aspects of protein fate including sub-cellular localization, stability, interaction partners, and function. It is known in coarse outline that ubiquitination regulates functional expression of KCNQ1 and HERG channels. However, the full scope and mechanistic bases of ubiquitin regulation of these channels, and the potential contributions of this posttranslational modification to LQTS are not known. There are several formidable obstacles to progress on these fronts owing to: diversity in the E2 ubiquitin conjugating, E3 ubiquitin ligase, and deubiquitination (DUB) enzymes; promiscuity among E3 ligase/substrate and DUB/substrate interactions; intrinsic complexity of the ubiquitin code (monoubiquitination vs polyubiquitiation; distinctive possible polyubiquitin chain linkages with different degradative and non-degradative signaling functions); and lack of spatio-temporal control over ubiquitination of specific substrates. This proposal is founded on exciting preliminary data in which we have circumvented the above complications by engineering methods to selectively target specific E3 ligases or DUBs to tagged KCNQ1 and HERG, respectively. Current dogma in the ubiquitin field holds that K48 ubiquitin chains are degradative while K63 chains have non-degradative signaling functions. Remarkably, our preliminary results enabled by the novel approaches indicate the exact opposite is true for KCNQ1 and HERG, possibly revealing a fundamental difference between cytosolic and membrane proteins. Our preliminary results further suggest that aberrant ubiquitination may underlie KCNQ1/HERG trafficking deficits in some LQT1/LQT2 mutations, and that this pathway may be targeted to rectify underlying abnormalities. Our long term objective is to elucidate molecular mechanisms controlling the surface density and functional regulation of KCNQ1 and HERG channels in heart under both physiological and pathological conditions, and to bridge the mechanistic insights to advance personalized therapy for LQTS and life- threatening cardiac arrhythmias. We combine state-of-the-art, innovative approaches: develop engineered E3 ligases/DUBs to enable unprecedented spatio-temporal control of KCNQ1/HERG ubiquitination; high- throughput flow cytometry; proteomics; biochemistry; and electrophysiology to address three specific aims: 1) Develop and utilize engineered E3 ligases to control spatiotemporal, linkage-specific ubiquitination of KCNQ1 and HERG, and to elucidate the ubiquitin code regulation of these channels. 2) Develop and utilize engineered deubiquitinases to control spatiotemporal, linkage-specific deubiquitination of KCNQ1 and HERG to elucidate ubiquitin code regulation of these channels. 3) Determine role of aberrant ubiquitination in diverse trafficking- deficient LQT1/LQT2 mutations, and assess opportunities for rescue.